# Patent

## Title
Production of 1,3-dienes by enzymatic conversion of 3-hydroxyalk 4-enoates and/or 3-phosphonoxyalk-4-enoates

## Abstract

The present invention relates to a method for generating 1,3-diene compounds through a biological process. More specifically, the invention relates to a method for producing 1,3-diene compounds (for example butadiene or isoprene) from molecules of the 3-hydroxyalk-4-enoate type or from 3-phosphonoxyalk-4-enoates.

## Background
This Application is a 371 National Phase filing of EP 2012075921 filed Dec. 18, 2012, which is a continuation of EP 11 194 704 which was filed on Dec. 20, 2011 and a nonprovisional of U.S. Ser. No. 61/578,058 filed Dec. 20, 2011, which are all incorporated by reference in their entirety.


The present invention relates to a method for generating 1,3-dienes through a biological process. More specifically, the invention relates to a method for producing 1,3-dienes (for example 1,3-butadiene or 2-methyl-1,3-butadiene (isoprene)) from molecules of the 3-hydroxyalk-4-enoate type or from 3-phosphonoxyalk-4-enoates.
1,3-dienes such as 1,3-butadiene or 2-methyl-1,3-butadiene (isoprene) are important molecules for the industry. Isoprene, for example, is a key compound for the tire industry, and also has many applications in the adhesives. It is produced chemically using several routes:

    
    
        Extractive distillation from oil (C5 cut)
        Dehydrogenation of iso-amylene
        Double dehydrogenation of isopentane
        Reaction of isobutene and formaldehyde
        Reaction of acetone and acetylene
        Propylene dimerization
    
    


WO 2009/076676 reports a metabolic pathway to isoprene. The pathway is based on the dephosphorylation-dehydration of downstream intermediates in the mevalonate pathway, i.e. isoprenyl-pyrophosphate or prenyl-pyrophosphate. This process has the drawback of requiring going through the whole mevalonate pathway: double phosphorylation of mevalonate, followed by a decarboxylation-dehydration into isoprenyl-pyrophosphate, further isomerised into prenyl-pyrophosphate, and finally double dephosphorylation/dehydration into isoprene.
Butadiene (1,3-butadiene) is a conjugated diene with the formula C4H6. It is an important industrial chemical used as a monomer in the production of synthetic rubber, nylon, ABS (Acrylonitrile-butadiene-styrene), plastics, latex. There exist different possibilities to produce butadiene. Butadiene is, for example, produced as a by product of the steam cracking process used to produce ethylene and other olefins. In this process butadiene occurs in the C4 stream and is normally isolated from other byproducts by extraction into a polar aprotic solvent, such as acetonitrile, from which it is then stripped. Butadiene can also be produced by the catalytic dehydrogenation of normal butane or it can be produced from ethanol. In the latter case, two different processes are in use. In a single-step process, ethanol is converted to butadiene, hydrogen and water at 400-450° C. over a metal oxide catalyst (Kirshenbaum, I. (1978), Butadiene. In M. Grayson (Ed.), Encyclopedia of Chemical Technology, 3rd ed., vol. 4, pp. 313-337. New York: John Wiley & Sons). In a two-step process, ethanol is oxidized to acetaldehyde which reacts with additional ethanol over a tantalum-promoted porous silica catalyst at 325-350° C. to yield butadiene (Kirshenbaum, I. (1978), loc cit.). Butadiene can also be produced by catalytic dehydrogenation of normal butenes. WO2012/018624 (US2012/0021478) proposed on a theoretical level various pathways for the enzymatic production of 1,3-butadiene including a pathway involving the decarboxylation of 3-hydroxypent-4-enoate.
For the past two decades, genetic engineering technologies have made possible the modification of the metabolism of micro-organisms, and hence their use to produce key substances which they would otherwise produce at a low yield. By enhancing naturally occurring metabolic pathways, these technologies open up new ways to bio-produce numerous compounds of industrial relevance. Several industrial compounds such as amino-acids for animal feed, biodegradable plastics or textile fibres are now routinely produced using genetically modified organisms.
There is still a need to provide environmentally friendly, cost efficient and simple methods for producing the above-mentioned compounds.
The present application addresses this need by the provision of the embodiments as specified in the claims.
The present invention is based on the design of a novel synthetic pathway for the synthesis of 1,3-diene compounds based on the conversion of 3-hydroxyalk-4-enoates and 3-phosphonoxyalk-4-enoates. The invention is based on the demonstration that said conversion can be carried out biologically, by using an enzyme catalyzing a decarboxylase reaction. The invention can be implemented in vitro, in cell-free systems, or by using organisms, in particular microorganisms. The invention also relates to the production of 1,3-diene compounds from a carbon source, and particularly a carbohydrate (in particular glucose), a polyol (in particular glycerol), a biodegradable polymer (in particular starch, cellulose, poly-3-hydroxyalkenoate) the carbon source being converted by a microorganism to a metabolic intermediate belonging to the 3-hydroxyalk-4-enoate family, which is then converted to 1,3-diene compound.
More specifically, the invention relates to a method for producing a 1,3-diene compound characterized in that it comprises a step of converting a 3-hydroxyalk-4-enoate in the presence of an enzyme having decarboxylase activity into a 1,3-diene compound. Thus, the method comprises the enzymatically catalyzed decarboxylation of a 3-hydroxyalk-4-enoate.
The term “3-hydroxyalk-4-enoate”, as used herein, denotes a molecule which responds to the following general formula:

Cn+1H2nO3 

with 3<n<7, and comprising 3-hydroxypent-4-enoate as common motif (FIG. 1B) and optionally a methyl substitution on carbon 3 and carbon 4.

In a preferred embodiment, “3-hydroxyalk-4-enoate”, as used herein, denotes a molecule responding to the following structural formula:

HO—CO—CH2—C(R1)(OH)—C(R2)═CH2 or O−—CO—CH2—C(R1)(OH)—C(R2)═CH2,

wherein R1 and R2 are selected, independently, from the group consisting of a hydrogen atom and a methyl group.

Carbon 3 is a chiral (stereogenic) center. The present definition encompasses the two chiral forms, even if one of the two forms, for example the R form, is the main form produced naturally. The suffix “oate”, as used herein, can interchangeably denote either the carboxylate ion (COO—) or carboxylic acid (COOH). It is not used to denote an ester.
The term “diene” (or diolefin) as used herein denotes a hydrocarbon that contains two conjugated carbon double bonds, with a general formula of CnH2n−2, where n is an integer with 3<n<7, i.e. n can be 4, 5 or 6.
The term “1,3-diene”, as used herein, denotes a molecule responding to the following structural formula H2C═C(R1)—C(R2)═CH2, wherein R1 and R2 are selected, independently, from the group consisting of a hydrogen atom and a methyl group.
In one particular embodiment the 3-hydroxyalk-4-enoate converted according to the method of the present invention is 3-hydroxypent-4-enoate and the resulting 1,3-diene compound is 1,3-butadiene.
In another embodiment the 3-hydroxyalk-4-enoate converted according to the method of the present invention is 3-hydroxy-4-methylpent-4-enoate or 3-hydroxy-3-methylpent-4-enoate and the resulting 1,3-diene compound is isoprene.
The term “enzyme having a decarboxylase activity” in the context of the present invention refers to an enzyme which is capable of decarboxylating a 3-hydroxyalk-4-enoate so as to lead to a 1,3-diene compound.
In one embodiment the enzyme having the activity of a decarboxylase is an enzyme which can be or is classified as a diphosphomevalonate decarboxylase or is an enzyme which is derived from such an enzyme and which has the capacity to decarboxylate a 3-hydroxyalk-4-enoate so as to produce a 1,3-diene compound. Diphosphomevalonate decarboxylase is classified with the EC number EC 4.1.1.33. A diphosphomevalonate decarboxylase is naturally able to catalyze the decarboxylation of mevalonate diphosphate. In this reaction ATP and 5-diphosphomevalonate are converted into ADP, phosphate, isopentenyl diphosphate and CO2. The reaction catalyzed by a diphosphomevalonate decarboxylase is shown in FIG. 1A. The activity of a diphosphomevalonate decarboxylase can be measured according to methods known in the art, e.g. in Reardon et al. (Biochemistry 26 (1987), 4717-4722). Preferably, the activity is measured by the spectrophotometric assay as described in Cardemil and Jabalquinto (Methods Enzymol. 110 (1985), 86-92). In this case, the reaction mixture (1 ml final volume) contains 0.1 M Tris-HCl buffer, pH 7.0, 0.1 M KCl, 5 mM ATP, 6 mM MgCl2, 0.5 mM phosphoenolpyruvate, 0.23 mM NADH, 6.5 units of pyruvate kinase, 11.8 units of lactate dehydrogenase, mevalonate 5-diphosphate decarboxylase, and 0.15 mM mevalonate 5-pyrophosphate is added to start the reaction. The assay is performed at 30° C. in a thermostatted spectrophotometer.
It has been reported that at least in some cases the reaction catalyzed by diphosphomevalonate decarboxylase is divalent cation-dependent (see, e.g., Krepkiy et al., Protein Science 13 (2004), 1875-1881; Michihara et al., Biol. Pharm. Bull. 25 (2002), 302-306).
Diphosphomevalonate decarboxylase is an enzyme which, in its natural function, is part of the mevalonate pathway for isoprenoid synthesis in bacteria and of the sterol biosynthesis pathway in eukaryotes. It has been identified and isolated from various organisms such as animals, fungi, yeasts and bacteria. It is also expressed by certain plants.
The three-dimensional structure of several diphosphomevalonate decarboxylases has already been determined (see, e.g., Byres et al. (J. Mol. Biol. 371 (2007), 540-553); Bonanno et al. (Proc. Natl Acad. Sci. USA 98 (2001), 12896-12901); Voynova et al., Archives of Biochemistry and Biophysics 480 (2008), 58-67)) and considerable knowledge is available about its active site, amino acid residues crucial for the catalytic reaction and the actual enzymatic reaction (see, e.g. Byres et al. (J. Mol. Biol. 371 (2007), 540-553); Bonanno et al. (Proc. Natl Acad. Sci. USA 98 (2001), 12896-12901)). In most cases the enzyme is composed of about 300 to 400 amino acids and uses ATP as co-substrate which is converted during the decarboxylation reaction into ADP and inorganic phosphate.
Diphosphomevalonate decarboxylases have been described for various organisms and also amino acid and nucleotide sequences encoding them are available for numerous sources.
In principle any diphosphomevalonate decarboxylase can be used in the context of the present invention, in particular from prokaryotic or eukaryotic organisms. Eukaryotic diphosphomevalonate decarboxylases are described, for example, for animals such as Rattus norvegicus, Gallus gallus, Homo sapiens, Mus musculus, Sus scrofa, D. melanogaster, C. elegans and Trypanosoma brucei, for plants such as Arabidopsis thaliana, Ginko biloba, Oryza sativa, Pisum sativum, for yeasts, such as Saccharomyces cerevisiae and Candida albicans. Also numerous prokaryotic diphosphomevalonate decarboxylases have been described, e.g. for Helicobacter, Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecium, Listeria monocytgenes, Leuconostoc citreum, Lactobacillus reuteri, to name just some. Table 1 provides a list of sequences of diphosphomevalonate decarboxylases from different organisms indicating the accession numbers under which they can be retrieved from the respective databases.








TABLE 1






Uniprot Accession


Organism
number







Bombyx mori
A5A7A2


Saccharomyces cerevisiae strain YJM7
A6ZSB7


Solanum lycopersicum
A8WBX7


Hevea brasiliensis
A9ZN03


Nicotiana langsdorffii × Nicotiana sanderae
B3F8H5


Saccharomyces cerevisiae (strain RM11-1a)
B3LPK0


Phaeodactylum tricornutum CCAP 1055
B7S422


Candida dubliniensis
B9W6G7


Pichia pastoris
C4QX63


Ashbya gossypii
Q751D8


Bos taurus
Q0P570


Danio rerio
Q5U403


Debaryomyces hanseni
Q6BY07


Dictyostelium discoideum
Q54YQ9


Homo sapiens
P53602


Mus musculus
Q99JF5


Rattus norvegicus
Q62967


Schizosaccharomyces pombe
O13963


Saccharomyces cerevisiae
P32377


Arnebia euchroma
Q09RL4


Aspergillus oryzae
Q2UGF4


Mus musculus
Q3UYC1


Ginkgo biloba
Q5UCT8


Rattus norvegicus
Q642E5


Oryza sativa subsp. japonica
Q6ETS8


Arabidopsis thaliana
Q8LB37


Encephalitozoon cuniculi
Q8SRR7


Hevea brasiliensis
Q944G0









The sequences mentioned in Table 1 are those available in UniProt Release 2011_12 (from uniprot.org/downloads).
Examples of diphosphomevalonate decarboxylases from different organisms are given in SEQ ID NO: 1 to 19 and 22 to 29. In a preferred embodiment of the present invention the diphosphomevalonate decarboxylase is an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 19 and 22 to 29 or a sequence which is at least n % identical to any of SEQ ID NO: 1 to 19 or 22 to 29 and having the activity of a diphosphomevalonate decarboxylase with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. Particularly preferred are sequences from bacteria of the genus Thermoplasma, Picrophilus, Ferroplasma or Streptococcus, and most preferred are sequences of the species Thermoplasma acidophilum (see, e.g., SEQ ID NO: 18), Thermoplasma volcanicum (see, e.g., SEQ ID NO: 17), Picrophilus torridus (e.g. strain DSM 9790; see, for example, SEQ ID NOs: 6, 16, 20 and 21), Ferroplasma acidarmanus (e.g. F. acidarmanus fer1; see, for example, SEQ ID NO: 19), Streptococcus mitis (e.g. strain B6; see, for example, SEQ ID NO: 24), Streptococcus infantarius (e.g. S. infantarius subsp infantarius ATCC BA-201; see, for example, SEQ ID NO:23), S. gallolyticus (see, e.g., SEQ ID NO:25), Streptococcus sp. M134 (see, e.g., SEQ ID NO: 27), S. salivarius (e.g. SK126; see, for example, SEQ ID NO:29), S. suis (e.g. S. suis 89/1591; see, for example, SEQ ID NO: 28), S. sanguinis (e.g., SK36; see, for example, SEQ ID NO: 26) or S. gordonii (see, e.g., SEQ ID NO:12).
Preferably, the degree of identity is determined by comparing the respective sequence with the amino acid sequence of any one of the above-mentioned SEQ ID NOs. When the sequences which are compared do not have the same length, the degree of identity preferably either refers to the percentage of amino acid residues in the shorter sequence which are identical to amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer sequence which are identical to amino acid residues in the shorter sequence. The degree of sequence identity can be determined according to methods well known in the art using preferably suitable computer algorithms such as CLUSTAL.
When using the Clustal analysis method to determine whether a particular sequence is, for instance, 80% identical to a reference sequence default settings may be used or the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.
Preferably, the degree of identity is calculated over the complete length of the sequence.
Moreover, if the term “homology” is used in the context of the present invention, this term preferably means “sequence identity”.
In a preferred embodiment the decarboxylase employed in the method according to the invention is a diphosphomevalonate decarboxylase from Picrophilus torridus or an organism which is evolutionary closely related to Picrophilus torridus. In a further preferred embodiment the decarboxylase originates from an organism of the genus Picrophilus, Thermoplasma or Ferroplasma, more preferably of the species Picrophilus torridus, Picrophilus oshimae, Thermoplasma volcanicum, Thermoplasma acidophilum, Ferroplasma acidarmanus or Ferroplasma cupricumulans. In another embodiment the decarboxylase originates from an organism of the genus Streptococcus, preferably of the species Streptococcus mitis, Streptococcus infantarius, S. gallolyticus, Streptococcus sp. M134, S. salivarius, S. suis, S. sanguinis or S. gordonii. 
Particularly preferred are decarboxylases from Thermoplasma acidophilum and from Streptococcus mitis. 
In a particularly preferred embodiment the decarboxylase employed in the method according to the invention is a diphosphomevalonate decarboxylase which comprises the amino acid sequence as depicted in SEQ ID NO: 6, 16, 17, 18 or 19 or which comprises an amino acid sequence which is at least n % identical to any of SEQ ID NO: 6, 16, 17, 18 or 19 and which has the activity of a diphosphomevalonate decarboxylase with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. The enzyme showing the amino acid sequence as shown in SEQ ID NOs:6 and 16 originates from Picrophilus torridus. Further preferred decarboxylases to be employed in the method according to the present invention are diphosphomevalonate decarboxylases which originate from organisms which are phylogenetically closely related to Picrophilus torridus, such as other bacteria of the genus Picrophilus, such as Picrophilus oshimae, bacteria of the genus Ferroplasma, e.g. Ferroplasma acidarmanus (SEQ ID NO:19), or of the genus Thermoplasma, such as Thermoplasma acidophilum (SEQ ID NO:18) and Thermoplasma volcanium (SEQ ID NO:17). The diphosphomevalonate decarboxylase of Thermoplasma acidophilum (AC number Q9HIN1) shows a homology of 38% to SEQ ID NO:6 and that of Thermoplasma volcanium (AC number Q97BY2) shows a homology of about 42% to SEQ ID NO:6.
The sequence shown in SEQ ID NO: 18 represents an enzyme identified in Thermoplasma acidophilum. In Genbank this enzyme is classified as a mevalonate diphosphate decarboxylase. However, it is known from Chen and Poulter (Biochemistry 49 (2010), 207-217) that in Th. acidophilum there exists an alternative mevalonate pathway which involves the action of a mevalonate-5-monophosphate decarboxylase. Thus, it is possible that the enzyme represented by SEQ ID NO: 18 actually represents a mevalonate-5-monophosphate decarboxylase.
The same may hold true for other archae bacteria. Therefore, in another preferred embodiment the decarboxylase employed in method according to the present invention is a mevalonate-5-monophosphate decarboxylase. Such an enzyme is capable of converting mevalonate-5-monophosphate into isopentenyl monophosphate. This activity can be measured in the same manner as the activity of a mevalonate diphosphate decarboxylase described above with the exception that mevalonate-5-monophosphate is used as a substrate.
In a further particularly preferred embodiment the decarboxylase employed in the method according to the invention is a diphosphomevalonate decarboxylase which is encoded by a nucleotide sequence as shown in SEQ ID NO: 20 or 21 or by a nucleotide sequence which is at least n % identical to any of SEQ ID NO: 20 or 21 and which encodes an enzyme having the activity of a diphosphomevalonate decarboxylase with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99. SEQ ID NO: 20 is the native nucleotide sequence encoding the MDP decarboxylase from P. torridus including at the N-terminus a His-tag. SEQ ID NO: 21 is a codon optimized sequence coding for the MDP decarboxylase from P. torridus including at the N-terminus a His-tag.
The decarboxylase, preferably diphosphomevalonate decarboxylase or mevalonate-5-monophosphate decarboxylase, employed in the process according to the invention can be a naturally occurring decarboxylase or it can be a decarboxylase which is derived from a naturally occurring decarboxylase, e.g. by the introduction of mutations or other alterations which, e.g., alter or improve the enzymatic activity, the stability, etc.
The term “decarboxylase”, “diphosphomevalonate decarboxylase”, “mevalonate-5-monophosphate decarboxylase”, “a protein/enzyme having the activity of a decarboxylase” or “a protein/enzyme having the activity of a diphosphomevalonate decarboxylase” in the context of the present application also covers enzymes which are derived from a decarboxylase, preferably a diphosphomevalonate decarboxylase or a mevalonate-5-monophosphate decarboxylase, which are capable of catalyzing the decarboxylation of a 3-hydroxyalk-4-enoate but which only have a low affinity to their natural substrate, e.g. mevalonate diphosphate or a mevalonate-5-monophosphate, or do no longer accept their natural substrate. Such a modification of the preferred substrate allows to improve the conversion of a 3-hydroxyalk-4-enoate into a 1,3-diene compound and to reduce the production of the possibly occurring by-product isoprenyl pyrophosphate. Methods for modifying and/or improving the desired enzymatic activities of proteins are well-known to the person skilled in the art and include, e.g., random mutagenesis or site-directed mutagenesis and subsequent selection of enzymes having the desired properties or approaches of the so-called “directed evolution”, DNA shuffling or in vivo evolution.
For example, for genetic engineering in prokaryotic cells, a nucleic acid molecule encoding a decarboxylase can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, “primer repair”, restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods. The resulting decarboxylase variants are then tested for their enzymatic activity and in particular for their capacity to prefer a 3-hydroxy-4-enoate as a substrate rather than, e.g. mevalonate diphosphate or a mevalonate-5-monophosphate.
Such methods for identifying variants with improved enzymatic properties as regards the production of a 1,3-diene compound may also be carried out in the presence of a cofactor which allows for a steric and/or electronic complementation in the catalytic site of the enzyme due to the fact that the a 3-hydroxyalk-4-enoate substrate may be shorter than the natural substrate, e.g. mevalonate diphosphate in the case of diphosphomevalonate decarboxylase. Examples for such a cofactor would be phosphono-phosphate or phosphonamido-phosphate or orthophosphate.
The modified version of the decarboxylase accepting or preferring a 3-hydroxyalk-4-enoate as a substrate but having a low affinity to its natural substrate or no longer accepting its natural substrate may be derived from a naturally occurring decarboxylase or from an already modified, optimized or synthetically synthesized decarboxylase.
It is known that the conversion of mevalonate diphosphate into an isopentenyl diphosphate by a mevalonate diphosphate (MDP) decarboxylase (E.C. 4.1.1.33) takes place by the conversion of MDP into the corresponding 3-phosphonoxy compound which is then decarboxylated to lead to isopentenyl diphosphate. The reaction carried out by MDP decarboxylase using MDP as a substrate is depicted in FIG. 1A.
FIG. 1 B shows a scheme showing the conversion of a 3-hydroxyalk-4-enoate into a 1,3-diene compound using a mevalonate diphosphate decarboxylase. The intermediate in this case is a 3-phosphonoxyalk-4-enoate. FIG. 2 shows the conversion of 3-hydroxypent-4-enoate into 1,3-butadiene using a mevalonate diphosphate decarboxylase. The intermediate in this case is 3-phosphonoxypent-4-enoate. FIG. 3 shows the conversion of 3-hydroxy-4-methylpent-4-enoate and 3-hydroxy-4-methylpent-4-enoate into isoprene using a mevalonate diphosphate decarboxylase. The intermediate in this case is 3-phosphonoxy-4-methylpent-4-enoate or 3-phosphonoxy-3-methylpent-4-enoate, respectively.
It has been found that different decarboxylases, in particular mevalonate diphosphate decarboxylases, catalyze the two above mentioned steps with different efficiencies, i.e. that some decarboxylases catalyze the first step with a higher efficiency than other decarboxylases and that some decarboxylases show a preference for the second step, i.e. the decarboxylation step, and that therefore the efficiency of the whole reaction can be significantly increased by combining corresponding enzymes.
Thus, in another embodiment, the method according to the invention is characterized in that two types of enzymes are combined in order to increase the efficiency of the production rate. More specifically, the present invention relates to a method for producing a 1,3-diene compound, characterized in that it comprises the conversion of a 3-hydroxyalk-4-enoate into said 1,3-diene compound by

    (i) a first enzyme having an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate; and
    (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound by a decarboxylation reaction.


The term “3-hydroxyalk-4-enoate”, as used herein, refers to a compound as defined herein-above.
The term “3-phosphonoxyalk-4-enoate” denotes a molecule which responds to the following general formula:
The term “3-phosphonoxyalk-4-enoate” denotes a molecule which responds to the following general formula:

Cn+1H2n+1O6P,

with 3<n<7, and comprising 3-phosphonoxypent-4-enoate as common motif (FIG. 1B) and optionally a methyl substitution on carbon 3 and carbon 4.

In preferred embodiment, “3-phosphonoxyalk-4-enoate”, as used herein, denotes a molecule responding to the following structural formula:

HO—CO—CH2—C(R1)(O—PO3H2)—C(R2)═CH2 or O−—CO—CH2—C(R1)(O—PO3H2)—C(R2)═CH2,

wherein R1 and R2 are selected, independently, from the group consisting of a hydrogen atom and a methyl group.

A 3-phosphonoxyalk-4-enoate corresponds to the phosphate ester of the alcohol group in 3-hydroxyalk-4-enoate, as previously described. This phosphate group can be fully protonated or bear one or two negative charges as in the formulas:

HO—CO—CH2—C(R1)(O—PO3H2)—C(R2)═CH2 

HO—CO—CH2—C(R1)(O—PO3H−)—C(R2)═CH2 

HO—CO—CH2—C(R1)(O—PO32−)—C(R2)═CH2 

O−—CO—CH2—C(R1)(O—PO3H2)—C(R2)═CH2 

O−—CO—CH2—C(R1)(O—PO3H−)—C(R2)═CH2 

O−—CO—CH2—C(R1)(O—PO32)—C(R2)═CH2 

Carbon 3 is a chiral (stereogenic) center. The present definition encompasses the two chiral forms, even if one of the two forms, for example the R form, is the main form produced naturally. The suffix “oate”, as used herein, can interchangeably denote either the carboxylate ion (COO—) or carboxylic acid (COOH). It is not used to denote an ester.
The term “1,3-diene”, as used herein, refers to a compound as defined herein-above.
The term “an enzyme having an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate” means an enzyme which can phosphorylate a 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate. The phosphate group comes preferably from an ATP molecule.
This activity can, e.g., be measured as described in the attached Examples, in particular Examples 2, 3, 7 and 8. One possibility is, for example, to incubate the respective enzyme with the 3-hydroxyalk-4-enoate and ATP and to measure the production of ADP (which reflects the production of the corresponding 3-phosphonoxyalk-4-enoate). Assays for measuring the production of ADP are known to the person skilled in the art. One of these methods is the well known pyruvate kinase/lactate dehydrogenase assay. In this case the assay measures the rate of NADH absorbance decrease at 340 nm which is proportional to the ADP quantity. In a preferred embodiment the term “an enzyme having an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate” means an enzyme which can convert 3-hydroxypent-4-enoate and ATP into 3-phosphonoxypent-4-enoate and ADP or an enzyme which can convert 3-hydroxy-4-methylpent-4-enoate or 3-hydroxy-4-methylpent-4-enoate and ATP into 3-phosphonoxy-4-methylpent-4-enoate or 3-phosphonoxy-3-methylpent-4-enoate, respectively, and ADP. Even more preferably such an enzyme can catalyze the reaction of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate with a KM of 10 mM or lower, e.g. with a KM of 5 mM or lower, preferably of 1 mM or lower and even more preferably of 0.1 mM or lower. In a particularly preferred embodiment such an enzyme can catalyze the reaction of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate with a kcat of at least 0.05 s−1, preferably with a kcat of at least 0.09 s−1, particularly preferred with a kcat of at least 0.1 s−1, more preferred of at least 0.2 s−1 and even more preferred with a kcat of at least 1.0 s−1.
In a particularly preferred embodiment the capacity to convert a 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate, e.g. 3-hydroxypent-4-enoate and ATP into 3-phosphonoxyalk-4-enoate and ADP, is measured in an assay as described in Examples 2, 3, 7 or 8.
The term “an enzyme having an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound by a decarboxylation reaction” means an enzyme which can catalyze a reaction by which there is a decarboxylation and dephosporylation of the 3-phosphonoxyalk-4-enoate thereby leading to the corresponding 1,3-diene compound.
This activity can, e.g., be measured as described in the appended Examples, in particular in Examples 6 and 11. One possibility is thus to incubate the respective enzyme with the corresponding 3-phosphonoxyalk-4-enoate under conditions which in principle allow the decarboxylation and the dephosphorylation and to detect the production of the corresponding 1,3-diene compound, e.g. by gas chromatography. In a preferred embodiment the term “an enzyme having an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound” means an enzyme which can convert 3-phosphonoxypent-4-enoate into 1,3-butadiene or an enzyme which can convert 3-phosphonoxy-4-methylpent-4-enoate or 3-phosphonoxy-3-methylpent-4-enoate into isoprene, preferably under the conditions described in Example 6 in which 3-phosphonoxy-4-methylpent-4-enoate or 3-phosphonoxy-3-methylpent-4-enoate is used instead of 3-phosphonoxypent-4-enoate in case of the synthesis of isoprene. Even more preferably such an enzyme can catalyze the reaction of converting the 3-phosphonoxyalk-4-enoate into the corresponding 1,3-diene compound (via decarboxylation and dephosphorylation) with a KM of 100 mM or lower, e.g. with a KM of 75 mM or lower, or with a KM of 50 mM or lower, preferably of 10 mM or lower or 5 mM or lower or 1 mM or lower, and even more preferably of 0.1 mM or lower. In a particularly preferred embodiment such an enzyme can catalyze the reaction of converting the 3-phosphonoxyalk-4-enoate into the corresponding 1,3-diene compound with a kcat of at least 10−6 s−1, preferably with a kcat of at least 10−4 s−1, e.g. with a kcat of at least 10−3 s−1 or with a kcat of at least 10−2 s−1, such as with a kcat of at least 10−1 s−1, for example with a kcat of at least 0.2 s−1, preferably with a kcat of at least 0.5 s−1, particularly preferred with a kcat of at least 1.0 s−1, more preferred of at least 2.0 s−1 and even more preferred with a kcat of at least 5.0 s−1. In a particularly preferred embodiment the capacity to convert a 3-phosphonoxyalk-4-enoate into a 1,3-diene compound is measured in an assay as described in Example 6 or in Example 11.
In one preferred embodiment an enzyme mentioned in (i) and (ii), above, is an enzyme which is considered by NCBI or an equivalent engine as having a COG3407 domain.
In a preferred embodiment of the method according to the invention the first enzyme (i) having an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate is selected from the group consisting of

    (A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 16 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 16 and showing an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 16;
    (B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 17 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 17 and showing an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 17;
    (C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 18 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 18 and showing an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 18; and
    (D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 19 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 19 and showing an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 19.


SEQ ID NO: 16 shows the amino acid sequence of an enzyme from Picrophilus torridus DSM 9790 (GenBank accession number AAT43941; Swissprot/TrEMBL accession number Q6KZB1).
SEQ ID NO: 17 shows the amino acid sequence of an enzyme from Thermoplasma volcanium (GenBank accession number BAB59465; Swissprot/TrEMBL accession number Q97BY2).
SEQ ID NO: 18 shows the amino acid sequence of an enzyme from Thermoplasma acidophilum (GenBank accession number CAC12426; Swissprot/TrEMBL accession number Q9HIN1).
SEQ ID NO: 19 shows the amino acid sequence of an enzyme from Ferroplasma acidarmanus fer1 (GenBank accession number ZP_05571615).
In a further preferred embodiment of the method according to the invention the second enzyme (ii) having an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound is selected from the group consisting of

    (a) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 12 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 12;
    (b) a protein comprising the amino acid sequence as shown in SEQ ID NO: 22 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 22 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 22;
    (c) a protein comprising the amino acid sequence as shown in SEQ ID NO: 23 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 23 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 23;
    (d) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 1 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 1;
    (e) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 7 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 7;
    (f) a protein comprising the amino acid sequence as shown in SEQ ID NO: 24 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 24 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 24;
    (g) a protein comprising the amino acid sequence as shown in SEQ ID NO: 25 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 25 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 25;
    (h) a protein comprising the amino acid sequence as shown in SEQ ID NO: 26 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 26 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 26;
    (i) a protein comprising the amino acid sequence as shown in SEQ ID NO: 27 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 27 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 27;
    (j) a protein comprising the amino acid sequence as shown in SEQ ID NO: 28 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 28 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 28; and
    (k) a protein comprising the amino acid sequence as shown in SEQ ID NO: 29 or a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence shown in SEQ ID NO: 29 and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of the protein having the amino acid sequence shown in SEQ ID NO: 29.


SEQ ID NO: 12 shows the amino acid sequence of an enzyme cloned from Streptococcus gordonii. SEQ ID NO: 22 shows the amino acid sequence of an enzyme from Streptococcus gordonii str. Challis substr. CH1 (GenBank accession number AAT43941; Swissprot/TrEMBL accession number A8UU9). SEQ ID NO: 23 shows the amino acid sequence of an enzyme from Streptococcus infantarius subsp infantarius ATCC BAA-102 (GenBank accession number EDT48420.1; Swissprot/TrEMBL accession number B1SCG0). SEQ ID NO: 1 shows the amino acid sequence of an enzyme from Homo sapiens (GenBank accession number AAC50440.1; Swissprot/TrEMBL accession number P53602.1). SEQ ID NO: 7 shows the amino acid sequence of an enzyme from Lactobacillus delbrueckii (GenBank accession number CAI97800.1; Swissprot/TrEMBL accession number Q1GAB2). SEQ ID NO: 24 shows the amino acid sequence of an enzyme from Streptococcus mitis (strain B6) (GenBank accession number CBJ22986.1). SEQ ID NO: 25 shows the amino acid sequence of an enzyme from Streptococcus gallolyticus UCN34 (GenBank accession number CBI13757.1). SEQ ID NO: 26 shows the amino acid sequence of an enzyme from Streptococcus sanguinis SK36 (GenBank accession number ABN43791.1). SEQ ID NO: 27 shows the amino acid sequence of an enzyme from Streptococcus sp. M143 (GenBank accession number EFA24040.1). SEQ ID NO: 28 shows the amino acid sequence of an enzyme from Streptococcus suis 89/1591 (GenBank accession number EEF63672.1). SEQ ID NO: 29 shows the amino acid sequence of an enzyme from Streptococcus salivarius SK126 (GenBank accession number EEK09252).
In a preferred embodiment of the method according to the invention the first enzyme (i) is as defined in (A) above and the second enzyme (ii) is as defined in (a) or (b) mentioned above, even more preferably the second enzyme is as defined in (f), (g), (h), (i), (j) or (k) mentioned above.
In another preferred embodiment of the method according to the invention the second enzyme (ii) having an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound is selected from any one of the proteins listed in the following Table or from a protein comprising an amino acid sequence which is at least 15% identical to the amino acid sequence of such a protein and showing an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound which is at least as high as the corresponding activity of said protein.










TABLE 2







Organism
Ref sequence GenBank









Methanosarcina mazei
AAM31457.1



Methanocaldococcus jannaschii
AAB98390.1



Staphylococcus saprophyticus
BAE19266.1



Streptococcus agalactiae
EAO73731.1



Enterococcus faecalis
AAO80711.1



Flavobacterium johnsoniae
ABQ04421.1



Bdellovibrio bacteriovorus
CAE79505.1



Chloroflexus aurantiacus
A9WEU8.1



Legionella pneumophila
CAH13175.1



Listeria monocytogenes
EAL09343.1



Metallosphaera sedula
ABP95731.1



Staphylococcus epidermidis
AAO03959.1



Streptococcus thermophilus
AAV60266.1



Bacillus coagulans
EAY45229.1



Chloroflexus aggregans
EAV09355.1



Lactobacillus brevis
ABJ64001.1



Lactobacillus fermentum
BAG27529.1



Lactobacillus plantarum
CAD64155.1



Lactobacillus salivarius
ABD99494.1



Lactococcus lactis sp. lactis
AAK04503.1



Dichelobacter nodosus
ABQ14154.1



Flavobacterium psychrophilum
CAL42423.1



Streptococcus pneumoniae
EDT95457.1



Streptococcus pyogenes
AAT86835.1



Streptococcus suis
ABP91444.1



Staphylococcus haemolyticus
BAE05710.1



Streptococcus equi
ACG62435.1



Arabidopsis thaliana
AAC67348.1



Borrelia afzelii
ABH01961.1



Encephalitozoon cuniculi
CAD25409.1



Streptomyces sp.
BAB07791.1



Streptococcus agalactiae
EAO73731.1



Streptococcus uberis
CAR41735.1



Gallus gallus
XP_423130



Salmo salmar
ACI34234



Natromonas pharaonis
CAI48881.1



Haloarcula marismortui
AAV46412.1



Haloquadratum walsbyi
CAJ51653.1










The sequences mentioned in Table 2 are those available in: Genetic Sequence Data Bank, Dec. 15, 2011, NCBI-GenBank Flat File Release 187.0, Distribution Release Notes 146413798 loci, 135117731375 bases, from 146413798 reported sequences (see ncbi.nih.gov/genbank/gbrel.txt).
As mentioned above, not only the proteins having the specifically mentioned amino acid sequences listed in the respective SEQ ID NOs or in Table 2 can be used, but also proteins which are considered by NCBI or an equivalent engine as having a COG3407 domain and, more preferred, proteins the amino acid sequence of which shows a homology of at least 15% to the specifically mentioned amino acid sequence and which have a respective enzymatic activity at least as high as the activity of a protein having the specifically mentioned amino acid sequence. Preferred enzymes advantageously have at least x % homology, wherein x is selected from the group consisting of 20, 25, 20, 35, 40, 45, 50, 55 and 60. In a further preferred embodiment the enzyme has at least 65% sequence homology, preferably at least 70%, more preferably at least 75%, even more preferably, at least 80, 85, 90, 95, 96, 97, 98 or 99% homology to one of the sequences shown in any one of SEQ ID NO: 1 to 19 and 22 to 29, in particular, SEQ ID NOs: 1, 7, 12, 16, 17, 18, 19, 22, 23, 24, 25, 26, 27, 28 or 29 or to one of the sequences shown in Table 1. The percent of sequence homology can be determined by different methods and by means of software programs known to one of skill in the art, such as for example the CLUSTAL method or BLAST and derived software, or by using a sequence comparison algorithm such as that described by Needleman and Wunsch (J. Mol. Biol., 1970, 48:443) or Smith and Waterman (J. Mol. Biol., 1981, 147:195).
Such proteins showing the indicated degree of homology can, e.g., be other enzymes which occur naturally or which have been prepared synthetically. They include in particular enzymes which can be selected for their ability to produce alkenes according to the invention. Thus, a selection test comprises contacting the purified enzyme, or a microorganism producing the enzyme, with the substrate of the reaction and measuring the production of the respective compound, i.e. the 3-phosphonoxyalk-4-enoate or the 1,3-diene compound. Such selection tests can also be used to screen for enzymes with an optimized enzymatic activity for the substrate to be converted into the 3-phosphonoxyalk-4-enoate or the 1,3-diene compound, i.e. having an optimized activity with respect to one or more 3-hydroxyalk-4-enoates or 3-phosphonoxyalk-4-enoates.
Such screening methods are well-known in the art and include, e.g. protein engineering techniques such as random mutagenesis, massive mutagenesis, site-directed mutagenesis, DNA shuffling, synthetic shuffling, in vivo evolution, or complete synthesis of genes and subsequent screening for the desired enzymatic activity.
The enzyme used in the invention can thus be natural or synthetic, and produced by chemical, biological or genetic means. It can also be chemically modified, for example in order to improve its activity, resistance, specificity, purification, or to immobilize it on a support.
Enzymes which are able to catalyze the above described reactions for converting a 3-hydroxyalk-4-enoate into a 1,3-diene compound via a 3-phosphonoxyalk-4-enoate are often enzymes which can be classified in the phylogenetic superfamily of mevalonate diphosphate (MDP) decarboxylases (enzyme nomenclature EC 4.1.1.33).
Accordingly, in a preferred embodiment, the enzyme defined in (i) or (ii) above, is a MDP decarboxylase. In the context of the present invention a MDP decarboxylase is defined as an enzyme which can at least catalyze the conversion of 5-diphospho-3-phosphomevalonate into isopentenyl-5-diphosphate and CO2 or which can at least catalyze the reaction of converting mevalonate diphosphate and ATP into 5-diphospho-3-phosphomevalonate and ADP. Preferably, such an enzyme can catalyze both reactions.
In another preferred embodiment the enzyme defined in (i) above, is an enzyme as defined in (i) (C). The sequence shown in SEQ ID NO: 18 represents an enzyme identified in Thermoplasma acidophilum. In Genbank this enzyme is classified as a mevalonate diphosphate decarboxylase. However, it is known from Chen and Poulter (Biochemistry 49 (2010), 207-217) that in Th. acidophilum there exists an alternative mevalonate pathway which involves the action of a mevalonate-5-monophosphate decarboxylase. Thus, it is possible that the enzyme represented by SEQ ID NO: 18 actually represents a mevalonate-5-monophosphate decarboxylase. The same may hold true for other archae bacteria. Therefore, in another preferred embodiment the enzyme defined in (i) or (ii) above, is a mevalonate-5-monophosphate decarboxylase. Such an enzyme is capable of converting mevalonate-5-monophosphate into isopentenylmonophosphate.
In a further embodiment, the enzyme as defined in (ii) of any of the previously described embodiments is an enzyme which can be classified as a terpene synthase. The inventors were able to show that surprisingly a terpene synthase is able to catalyze the conversion of a 3-phosphonoxyalk-4-enoate into a 1,3-diene, in particular the conversion of 3-phosphonoxypent-4-enoate into 1,3-butadiene (see Example 11 and FIG. 8).
The terpene synthases constitute an enzyme family which comprises enzymes catalyzing the formation of numerous natural products always composed of carbon and hydrogen (terpenes) and sometimes also of oxygen or other elements (terpenoids). Terpenoids are structurally diverse and widely distributed molecules corresponding to well over 30000 defined natural compounds that have been identified from all kingdoms of life. In plants, the members of the terpene synthase family are responsible for the synthesis of the various terpene molecules from two isomeric 5-carbon precursor “building blocks”, isoprenyl diphosphate and prenyl diphosphate, leading to 5-carbon isoprene, 10-carbon monoterpene, 15-carbon sesquiterpene and 20-carbon diterpenes” (Chen et al.; The Plant Journal 66 (2011), 212-229).
The ability of terpene synthases to convert a prenyl diphosphate containing substrate to diverse products during different reaction cycles is one of the most unique traits of this enzyme class. The common key step for the biosynthesis of all terpenes is the reaction of terpene synthase on corresponding diphosphate esters. The general mechanism of this enzyme class induces the removal of the diphosphate group and the generation of an intermediate with carbocation as the first step. In the various terpene synthases, such intermediates further rearrange to generate the high number of terpene skeletons observed in nature. In particular, the resulting cationic intermediate undergoes a series of cyclizations, hydride shifts or other rearrangements until the reaction is terminated by proton loss or the addition of a nucleophile, in particular water for forming terpenoid alcohols (Degenhardt et al., Phytochemistry 70 (2009), 1621-1637).
The terpene synthases show a common catalytic mechanism which involves the formation of an allylic carbocation by the removal of a pyrophosphate leaving group, which evolves then towards various products (see the following scheme; Croteau, Chem. Rev. 87 (1987), 929-954; Croteau, Topics Curr. Chem. 209 (2000).
The different terpene synthases also share various structural features. These include a highly conserved C-terminal domain, which contains their catalytic site and an aspartate-rich DDXXD motif essential for the divalent metal ion (typically Mg2+ or Mn2+) assisted substrate binding in these enzymes (Green et al. Journal of biological chemistry, 284, 13, 8661-8669). In principle, any known enzyme which can be classified as belonging to the EC 4.2.3 enzyme superfamily can be employed.
In one embodiment of the present invention an isoprene synthase (EC 4.2.3.27) is used for the direct enzymatic conversion of a 3-phosphonoxyalk-4-enoate into a 1,3-diene. Isoprene synthase is an enzyme which catalyzes the following reaction:

Dimethylallyl diphosphateisoprene+diphosphate

This enzyme occurs in a number of organisms, in particular in plants and some bacteria. The occurrence of this enzyme has, e.g., been described for Arabidopsis thaliana, a number of Populus species like P. alba (UniProt accession numbers Q50L36, A9Q7C9, D8UY75 and D8UY76), P. nigra (UniProt accession number AOPFK2), P. canescence (UniProt accession number Q9AR86; see also Köksal et al., J. Mol. Biol. 402 (2010), 363-373), P. tremuloides, P. trichocarpa, in Quercus petraea, Quercus robur, Salix discolour, Pueraria montana (UniProt accession number Q6EJ97), Pueraria montana var. lobata (SEQ ID NO:30), Mucuna pruriens, Vitis vinifera, Embryophyta and Bacillus subtilis. In principle, any known isoprene synthase can be employed in the method according to the invention. In a preferred embodiment, the isoprene synthase employed in a method according to the present invention is an isoprene synthase from a plant of the genus Populus, more preferably from Populus trichocarpa or Populus alba. In another preferred embodiment the isoprene synthase employed in a method according to the present invention is an isoprene synthase from Pueraria montana, preferably from Pueraria montana var. lobata (UNIPROT: Q6EJ97), or from Vitis vinifera. Preferred isoprene synthases to be used in the context of the present invention are the isoprene synthase of Populus alba (Sasaki et al.; FEBS Letters 579 (2005), 2514-2518) or the isoprene synthases from Populus trichocarpa and Populus tremuloides which show very high sequence homology to the isoprene synthase from Populus alba. Another preferred isoprene synthase is the isoprene synthase from Pueraria montana var. lobata (kudzu) (Sharkey et al.; Plant Physiol. 137 (2005), 700-712; UNIPROT: Q6EJ97; SEQ ID NO:30).
In a preferred embodiment of the present invention the isoprene synthase is an enzyme comprising the amino acid sequence shown in SEQ ID NO: 30 or a sequence which is at least n % identical to SEQ ID NO: 30 and having the activity of an isoprene synthase with n being an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.
The activity of an isoprene synthase can be measured according to methods known in the art, e.g. as described in Silver and Fall (Plant Physiol (1991) 97, 1588-1591). In a typical assay, the enzyme is incubated with dimethylallyl diphosphate in the presence of the required co-factors, Mg2+ or Mn2+ and K+ in sealed vials. At appropriate time volatiles compound in the headspace are collected with a gas-tight syringe and analyzed for isoprene production by gas chromatography (GC).
Moreover, it is not only possible to use an isoprene synthase for converting a 3-phosphonoxyalk-4-enoate into a 1,3-diene according to the above shown scheme, but it is also possible to use other enzymes from the family of monoterpene synthases. Monoterpene synthases comprise a number of families to which specific EC numbers are allocated. However, they also include also a number of enzymes which are simply referred to as monoterpene synthases and which are not classified into a specific EC number. To the latter group belong, e.g., the monoterpene synthases of Eucalyptus globulus (UniProt accession number Q0PCI4) and of Melaleuca alternifolia described in Shelton et al. (Plant Physiol. Biochem. 42 (2004), 875-882). In particularly preferred embodiments of the present invention use is made of a monoterpene synthase of Eucalyptus globulus or of Melaleuca alternifolia. 
In other preferred embodiments of the method according to the invention the conversion of a 3-phosphonoxyalk-4-enoate into a 1,3-diene according to the above shown scheme is achieved by a terpene synthase belonging to one of the following families: alpha-farnesene synthases (EC 4.2.3.46), beta-farnesene synthases (EC 4.2.3.47), myrcene/(E)-beta-ocimene synthases (EC 4.2.3.15) and pinene synthase (EC 4.2.3.14).
Farnesene synthases are generally classified into two different groups, i.e. alpha-farnesene synthases (EC 4.2.3.46) and beta farnesene synthases (EC 4.2.3.47). Alpha-farnesene synthases (EC 4.2.3.46) naturally catalyze the following reaction:

(2E,6E)-farnesyl diphosphate(3E,6E)-alpha-farnesene+diphosphate

This enzyme occurs in a number of organisms, in particular in plants, for example in Malus×domestica (UniProt accession numbers Q84LB2, B2ZZ11, Q6Q2J2, Q6QWJ1 and Q32WI2), Populus trichocarpa, Arabidopsis thaliana (UniProt accession numbers A4FVP2 and P0CJ43), Cucumis melo (UniProt accession number B2KSJ5) and Actinidia deliciosa (UniProt accession number C7SHN9). In principle, any known alpha-farnesene synthase can be employed in the method according to the invention. In a preferred embodiment, the alpha-farnesene synthase employed in a method according to the present invention is an alpha-farnesene synthase from Malus×domestica (e.g. Seq ID NO:8), UniProt accession numbers Q84LB2, B2ZZ11, Q6Q2J2, Q6QWJ1 and Q32WI2; see also Green et al.; Photochemistry 68 (2007), 176-188).
Beta-farnesene synthases (EC 4.2.3.47) naturally catalyze the following reaction:

(2E,6E)-farnesyl diphosphate(E)-beta-farnesene+diphosphate

This enzyme occurs in a number of organisms, in particular in plants and in bacteria, for example in Artemisia annua (UniProt accession number Q4VM12), Citrus junos (UniProt accession number Q94JS8), Oryza sativa (UniProt accession number Q0J7R9), Pinus sylvestris (UniProt accession number D7PCH9), Zea diploperennis (UniProt accession number C7E5V9), Zea mays (UniProt accession numbers Q2NM15, C7E5V8 and C7E5V7), Zea perennis (UniProt accession number C7E5W0) and Streptococcus coelicolor (Zhao et al., J. Biol. Chem. 284 (2009), 36711-36719). In principle, any known beta-farnesene synthase can be employed in the method according to the invention. In a preferred embodiment, the beta-farnesene synthase employed in a method according to the present invention is a beta-farnesene synthase from Mentha piperita (Crock et al.; Proc. Natl. Acad. Sci. USA 94 (1997), 12833-12838).
Methods for the determination of farnesene synthase activity are known in the art and are described, for example, in Green et al. (Phytochemistry 68 (2007), 176-188). In a typical assay farnesene synthase is added to an assay buffer containing 50 mM BisTrisPropane (BTP) (pH 7.5), 10% (v/v) glycerol, 5 mM DTT. Tritiated farnesyl diphosphate and metal ions are added. Assays containing the protein are overlaid with 0.5 ml pentane and incubated for 1 h at 30° C. with gentle shaking. Following addition of 20 mM EDTA (final concentration) to stop enzymatic activity an aliquot of the pentane is removed for scintillation analysis. The olefin products are also analyzed by GC-MS.
Myrcene/(E)-beta-ocimene synthases (EC 4.2.3.15) are enzymes which naturally catalyze the following reaction:

Geranyl diphosphate(E)-beta-ocimene+diphosphate

or

Geranyl diphosphatemyrcene+diphosphate

These enzymes occur in a number of organisms, in particular in plants and animals, for example in Lotus japanicus (Arimura et al.; Plant Physiol. 135 (2004), 1976-1983), Phaseolus lunatus (UniProt accession number B1P189), Abies grandis, Arabidopsis thaliana (UniProt accession number Q9ZUH4), Actinidia chinensis, Vitis vinifera (E5GAG5), Perilla fructescens, Ochtodes secundiramea and in Ips pini (UniProt accession number Q58GE8). In principle, any known myrcene/ocimene synthase can be employed in the method according to the invention. In a preferred embodiment, the myrcene/ocimene synthase employed in a method according to the present invention is an (E)-beta-ocimene synthase from Vitis vinifera. 
The activity of an ocimene/myrcene synthase can be measured as described, for example, in Arimura et al. (Plant Physiology 135 (2004), 1976-1983). In a typical assay for determining the activity, the enzyme is placed in screwcapped glass test tube containing divalent metal ions, e.g. Mg2+ and/or Mn2+, and substrate, i.e. geranyl diphosphate. The aqueous layer is overlaid with pentane to trap volatile compounds. After incubation, the assay mixture is extracted with pentane a second time, both pentane fractions are pooled, concentrated and analyzed by gas chromatography to quantify ocimene/myrcene production.
Pinene synthase (EC 4.2.3.14) is an enzyme which naturally catalyzes the following reaction:

Geranyl diphosphatealpha-pinene+diphosphate

This enzyme occurs in a number of organisms, in particular in plants, for example in Abies grandis (UniProt accession number 0244475), Artemisia annua, Chamaecyparis formosensis (UniProt accession number C3RSF5), Salvia officinalis and Picea sitchensis (UniProt accession number Q6XDB5).
For the enzyme from Abies grandis a particular reaction was also observed (Schwab et al., Arch. Biochem. Biophys. 392 (2001), 123-136), namely the following:

6,7-dihydrogeranyl diphosphate6,7-dihydromyrcene+diphosphate

In principle, any known pinene synthase can be employed in the method according to the invention. In a preferred embodiment, the pinene synthase employed in a method according to the present invention is a pinene synthase from Abies grandis (UniProt accession number O244475; Schwab et al., Arch. Biochem. Biophys. 392 (2001), 123-136).
Methods for the determination of pinene synthase activity are known in the art and are described, for example, in Schwab et al. (Archives of Biochemistry and Biophysics 392 (2001), 123-136). In a typical assay, the assay mixture for pinene synthase consists of 2 ml assay buffer (50 mM Tris/HCl, pH 7.5, 500 mM KCl, 1 mM MnCl2, 5 mM dithiothreitol, 0.05% NaHSO3, and 10% glycerol) containing 1 mg of the purified protein. The reaction is initiated in a Teflon-sealed screw-capped vial by the addition of 300 mM substrate. Following incubation at 25° C. for variable periods (0.5-24 h), the mixture is extracted with 1 ml of diethyl ether. The biphasic mixture is vigorously mixed and then centrifuged to separate the phases. The organic extract is dried (MgSO4) and subjected to GC-MS and MDGC analysis.
According to the present invention it is also possible to employ in the present invention an enzyme which has been constructed by physically combining an enzyme as defined in (i), above, which is particularly efficient in catalyzing the conversion of the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate, with an enzyme as defined in (ii), above, which is particularly efficient in catalyzing the conversion of said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound by a decarboxylation reaction. This can be achieved, e.g., by fusing corresponding nucleic acids encoding the respective enzymes so as to produce a fusion protein or by mutating one enzyme so that it acquires a high efficiency for both catalytic activities.
The present invention also relates to the use of at least two enzymes, wherein one enzyme is selected from (i) as specified above and the other enzyme is selected from (ii) as specified above or of a microorganism producing said combination of enzymes, for producing a 1,3-diene compound from a 3-hydroxyalk-4-enoate.
The present invention also discloses organisms, preferably microorganisms, which produce at least two enzymes, wherein one enzyme is selected from (i) as specified above and the other enzyme is selected from (ii) as specified above.
The methods according to the invention can be carried out in vitro, in the presence of isolated enzymes (or enzyme systems additionally comprising one or more cofactors). In vitro preferably means in a cell-free system.
In one embodiment, the enzymes employed in the method are used in purified form to convert a 3-hydroxyalk-4-enoate or a 3-phosphonoxyalk-4-enoate to a 1,3-diene compound. However, such a method may be costly, since enzyme and substrate production and purification costs are high.
Thus, in another preferred embodiment, the enzymes employed in the method are present in the reaction as a non-purified extract, or else in the form of non-lysed bacteria, so as to economize on protein purification costs. However, the costs associated with such a method may still be quite high due to the costs of producing and purifying the substrates.
Accordingly, in one preferred embodiment, the enzymes, native or recombinant, purified or not, are used to convert a 3-hydroxyalk-4-enoate or a 3-phosphonoxyalk-4-enoate to a 1,3-diene compound. To do this, the enzymes are incubated in the presence of the substrate in physicochemical conditions allowing the enzymes to be active, and the incubation is allowed to proceed for a sufficient period of time. At the end of the incubation, one optionally measures the presence of the 1,3-diene compound by using any detection system known to one of skill in the art such as gas chromatography or colorimetric tests for measuring the formation of the 1,3-diene product, or of free phosphate, or else for measuring the disappearance of the 3-hydroxyalk-4-enoate substrate or of ATP or of the 3-phosphonoxyalk-4-enoate.
In a preferred embodiment, cofactors are added so as to best mimic the natural reaction or so as to provide steric or electronic complementation in the catalytic cleft. For example, if one of the enzymes used in the method according to the invention is an enzyme which naturally uses mevalonate disphosphate (MDP) as a substrate, the structure of 3-hydroxyalk-4-enoate leaves a large space in the catalytic cleft empty during enzyme-substrate binding since generally a 3-hydroxyalk-4-enoate corresponds to a fragment of MDP. Filling this space with a cofactor to replace the missing part of the substrate has the purpose of most closely mimicking the MDP molecule. As the cofactor is not modified during the reaction, it will therefore be added only in catalytic amounts. By chance, it may happen that the complementary cofactor of a reaction has a positive effect on the reaction of another substrate. Generally, the cofactor can be any molecule comprising a phosphoanhydride, and therefore having the general global formula R—PO2H—O—PO3H2, in which R is in particular H, a linear, branched or cyclic alkyl group, preferably having from 1 to 10 or from 1 to 5 carbon atoms, or any other monovalent organic group. The analogous motifs corresponding to methylene diphosphonate monoesters, having the general formula R—O—PO2H—CH2—PO3H2 in which phosphyanhydride is replaced by a methylene bridge having the advantage of not being hydrolyzed, are also part of the invention. More generally, the cofactors can be monophosphate, or even phosphate-free, analogs of the previous molecules, or else any other molecule that can improve the reaction yield by providing steric or electronic complementation in the enzyme catalytic site. The cofactor is advantageously selected between pyrophosphate ion and methyl diphosphate.
In a preferred embodiment, the conversion occurs in the presence of a co-substrate, said co-substrate preferably being a compound containing a phosphoanhydride, and preferably being ATP, an rNTP, a dNTP or a mixture of several of these molecules, a polyphosphate, or pyrophosphate. The co-substrate is generally present in the host. However, in another particular embodiment, a co-substrate can be added to the reaction, preferably selected from the group consisting of ATP, an rNTP, a dNTP, a mixture of several rNTPs or dNTPs, a polyphosphate, and preferably pyrophosphate, or a compound containing a phosphoanhydride (represented by the general formula X—PO3H2).
Although the decarboxylation step, i.e. the reaction defined as (ii) herein-above, does not require ATP consumption, it could be shown that the presence of ATP in the reaction could be beneficial. It is assumed that ATP might have an effect on the folding of the protein by the binding of ATP to the ATP-binding site of the diphosphomevalonate decarboxylase. In fact, this can be observed by eye: the purified enzyme has a tendency to precipitate, and the addition of ATP prevents this effect. It is considered that not only ATP but also other similar compounds like dATP, ADP, AMP or other NTPs or dNTPs have this effect. Thus, in a preferred embodiment, the method according to the present invention is carried with ATP, dATP, ADP, AMP or an NTP other than ATP or a dNTP as co-substrate.
In another preferred embodiment the method according to the invention is carried out in culture, in the presence of an organism, preferably a microorganism, producing the enzymes. Thus, in such an embodiment of the invention, an organism, preferably a microorganism, that produces the respective enzyme(s) is used. In a preferred embodiment, the (micro)organism is recombinant in that the enzyme(s) produced by the host are heterologous relative to the production host. The method can thus be carried out directly in the culture medium, without the need to separate or purify the enzymes. In an especially advantageous manner, a (micro)organism is used having the natural or artificial property of endogenously producing one or more 3-hydroxyalk-4-enoates and/or a 3-phosphonoxyalk-4-enoate, and also expressing or overexpressing the enzyme(s) as specified above, natural or modified, so as to produce 1,3-diene compounds directly from a carbon source present in solution.
For example, the method according to the invention can be carried out by using microorganisms which produce one or more 3-hydroxyalk-4-enoates. It has been, e.g., been described in Ulmer et al. (Macromolecules 27 (1994), 1675-1679) that Rhodospirillum rubrum is capable of producing polymers consisting of 3-hydroxypent-4-enoate when grown on 4-pentenoic acid or on an equimolar mixture of 4-pentenoic acid and pentanoic acid.
Moreover, it has been reported by Rodrigues et al. (Appl. Micobiol. Biotechnol. 43 (1995), 880-886 and Appl. Microbiol. Biotechnol. 53 (2000), 453-460) that certain strains of Burkholderia sp. show the capacity of accumulating 3-hydroxypent-4-enoic acid when supplied with glucose or gluconate as the sole carbon and energy source. Thus, in one embodiment of the production of 1,3-butadiene according to the present invention it is preferred to use a microorganism which is capable of producing 3-hydroxy-pentenoic acid, such as Rhodospirillum rubrum or Burkholderia sp. and which has been genetically engineered such that they overexpress the decarboxylase enzyme(s), said enzyme(s) preferably originating from an organism different from the host microorganism. The genetic modification can consist, e.g. in integrating the corresponding gene(s) encoding the enzyme(s) into the chromosome, expressing the enzyme(s) from a plasmid containing a promoter upstream of the enzyme-coding sequence, the promoter and coding sequence preferably originating from different organisms, or any other method known to one of skill in the art. Alternatively, other bacteria or yeasts may have specific advantages and can be chosen. For instance, a yeast such as Saccharomyces cerevisiae, an extremophilic bacterium such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae, microalgae, or photosynthetic bacteria can be used.
It is also conceivable to isolate the genes encoding the proteins which are responsible for the synthesis of 3-hydroxypent-4-enoic acid from, e.g., Rhodospirillum rubrum or Burkholderia sp. and to introduce these genes into another organisms, in particular a microorganism, such as e.g. E. coli or Saccharomyces, an extremophilic bacterium such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae, microalgae, or photosynthetic bacteria.
The organisms used in the invention can be prokaryotes or eukaryotes, preferably, they are microorganisms such as bacteria, yeasts, fungi or molds, or plant cells or animal cells. In a particular embodiment, the microorganisms are bacteria, preferably of the genus Escherichia, even more preferably of the species Escherichia coli. 
In another preferred embodiment, the microorganisms are recombinant bacteria of the genus Escherichia, preferably of the species Escherichia coli, having been modified so as to endogenously produce one or more 3-hydroxyalk-4-enoates, and converting them to 1,3-diene compounds.
In a further preferred embodiment the microorganism is a fungus, more preferably a fungus of the genus Saccharomyces, Schizosaccharomyces, Aspergillus or Trichoderma and even more preferably of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus niger or of the species Trichoderma reesei. In a particularly preferred embodiment the microorganism is a recombinant yeast producing 3-hydroxyalk-4-enoates and converting them to 1,3-diene compounds due to the expression of the enzymes specified above.
In another preferred embodiment, the method according to the invention makes use of a photosynthetic microorganism expressing the enzymes as specified above. Preferably, the microorganism is a photosynthetic bacterium, or a microalgae. Even more preferably such a microorganism has the natural or artificial property of endogenously producing one or more 3-hydroxyalk-4-enoates. In this case the microorganism would be capable of producing 1,3-diene compounds directly from CO2 present in solution.
It is also conceivable to use in the method according to the invention one microorganism that produces an enzyme as defined in (i) above and another microorganism which produces an enzyme as defined in (ii) above. Moreover, in a further embodiment at least one of the microorganisms is capable of producing one or more 3-hydroxyalk-4-enoates or, in an alternative embodiment, a further microorganism is used in the method which is capable of producing one or more 3-hydroxyalk-4-enoates.
In another preferred embodiment the method according to the invention makes use of a multicellular organism expressing the enzymes as defined above. Examples for such organisms are plants or animals.
In a particular embodiment, the method involves culturing microorganisms in standard culture conditions (30-37° C. at 1 atm, in a fermenter allowing aerobic growth of the bacteria) or non-standard conditions (higher temperature to correspond to the culture conditions of thermophilic organisms, for example).
In a further preferred embodiment the method of the invention is carried out in microaerophilic conditions. This means that the quantity of injected air is limiting so as to minimize residual oxygen concentrations in the gaseous effluents containing the 1,3-diene compound.
In another preferred embodiment the method according to the invention furthermore comprises the step of collecting gaseous 1,3-diene compounds degassing out of the reaction, i.e. recovering the products which degas, e.g., out of the culture. Thus in a preferred embodiment, the method is carried out in the presence of a system for collecting the 1,3-diene compound under gaseous form during the reaction.
As a matter of fact, short 1,3-diene compounds, and particularly butadiene, adopt the gaseous state at room temperature and atmospheric pressure. The method according to the invention therefore does not require extraction of the product from the liquid culture medium, a step which is always very costly when performed at industrial scale. The evacuation and storage of the gaseous hydrocarbons and their possible subsequent physical separation and chemical conversion can be performed according to any method known to one of skill in the art.
In a particular embodiment, the method also comprises detecting the 1,3-diene compound (for example butadiene or isoprene) which is present in the gaseous phase. The presence of the compound to be produced in an environment of air or another gas, even in small amounts, can be detected by using various techniques and in particular by using gas chromatography systems with infrared or flame ionization detection, or by coupling with mass spectrometry.
The present invention also relates to a method for producing a 1,3-diene compound comprising the step of enzymatically converting a 3-phosphonoxyalk-4-enoate into the corresponding 1,3-diene compound by use of an enzyme which can catalyze the conversion via decarboxylation and dephosphorylation.
As regards the preferred enzyme to be used in such a method, the same applies as has been set forth above in connection with (ii) of the method according to the invention as described herein-above.
Moreover, also with respect to the other preferred embodiments described above for the method according to the invention, the same applies to the method for producing a 1,3-diene compound from a 3-phosphonoxyalk-4-enoate.
Thus, the present invention in particular also relates to a method for the production of a 1,3-diene compound characterized in that it comprises the step of converting a 3-phosphonoxyalk-4-enoate with an enzyme having the activity of a terpene synthase into a 1,3-diene compound.
As mentioned above, the inventors have surprisingly found that a terpene synthase is able to catalyze the conversion of a 3-phosphonoxyalk-4-enoate into a 1,3-diene compound (see Example 11 and FIG. 8). The terms “3-phosphonoxyalk-4-enoate” and “1,3-diene” have the same meaning as described herein above and the same applies here as has been described above in connection with preferred embodiments. Thus, in one preferred embodiment the 3-phosphonoxyalk-4-enoate is 3-phosphonoxypent-4-enoate and the produced 1,3-diene is 1,3-butadiene. In another preferred embodiment, the 3-phosphonoxyalk-4-enoate is 3-phosphonoxy-4-methylpent-4-enoate or 3-phosphonoxy-3-methylpent-4-enoate and the 1,3-diene is isoprene.
As regards the terpene synthase to be employed in such a method and the corresponding preferred embodiments, the same applies as has been described herein above.
The present invention also relates to the use of organisms, preferably microorganisms, which produce the above described enzymes, preferably at least two enzymes, wherein one enzyme is selected from (i) as specified above and the other enzyme is selected from (ii) as specified above, for the production of a 1,3-diene compound from a 3-hydroxyalk-4-enoate. In a preferred embodiment such an organism is a recombinant organism in the sense that it is genetically modified due to the introduction of at least one nucleic acid molecule encoding at least one of the above mentioned enzymes. Preferably such a nucleic acid molecule is heterologous with regard to the organism which means that it does not naturally occur in said organism.
In a preferred embodiment such an organism is an organism which produces one or more 3-hydroxyalk-4-enoates. Rhodospirillum rubrum is, for example, capable of producing polymers consisting of 3-hydroxypent-4-enoate when grown on 4-pentenoic acid or on an equimolar mixture of 4-pentenoic acid and pentanoic acid. Moreover, certain strains of Burkholderia sp. show the capacity of accumulating 3-hydroxypent-4-enoic acid when supplied with glucose or gluconate as the sole carbon and energy source.
Thus, in one embodiment a microorganism is used which is capable of producing 3-hydroxy-pentenoic acid, such as Rhodospirillum rubrum or Burkholderia sp. and which has been genetically engineered such that it overexpresses the decarboxylase enzyme(s), said enzyme(s) preferably originating from an organism different from the host microorganism. It is also conceivable to isolate the genes encoding the proteins which are responsible for the synthesis of 3-hydroxypent-4-enoic acid from, e.g., Rhodospirillum rubrum or Burkholderia sp. and to introduce these genes into another organisms, in particular a microorganism, such as e.g. E. coli or Saccharomyces, an extremophilic bacterium such as Thermus thermophilus, or anaerobic bacteria from the family Clostridiae, microalgae, or photosynthetic bacteria.
Thus, the present invention also relates to the use of such an organism, preferably a microorganism, which comprises a nucleic acid molecule coding for an enzyme as defined in (i) above and which comprises a nucleic acid molecule coding for an enzyme as defined in (ii) above, for the production of a 1,3-diene compound from a 3-hydroxyalk-4-enoate. In a preferred embodiment at least one of the nucleic acid molecules is heterologous to the organism which means that it does not naturally occur in said organism. The microorganism is preferably a bacterium, a yeast or a fungus. In another preferred embodiment the organism is a plant or non-human animal. In a further preferred embodiment, the organism is an organism which produces one or more 3-hydroxyalk-4-enoates. As regards other preferred embodiments, the same applies as has been set forth above in connection with the method according to the invention.
Moreover, the present invention also relates to a composition comprising a microorganism as defined herein above, a suitable culture medium and a 3-hydroxyalk-4-enoate compound or a carbon source that can be converted by the microorganism to a 3-hydroxyalk-4-enoate compound.
The present invention also relates to the use of an enzyme having decarboxylase activity as described herein-above or of a combination of at least two enzymes, wherein one enzyme is selected from the following (i) and the other enzyme is selected from the following (ii) or of an organism, preferably a microorganism, as described herein-above or of a composition according to the invention, for producing a 1,3-diene compound from a 3-phosphonoxyalk-4-enoate, wherein (i) and (ii) are as follows:

    (i) a first enzyme having an activity of converting the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate; and
    (ii) a second enzyme being different from the first enzyme and having an activity of converting said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound by a decarboxylation reaction.


As regards the preferred embodiments of the different components recited, the same applies as has been set forth above in connection with the method according to the invention.
The present invention also relates to the use of a terpene synthase for producing a 1,3-diene compound from a 3-phosponoxyalk-4-enoate by the dephosphorylation-decarboxylation of the 3-phosphonoxyalk-4-enoate.



FIG. 1 shows the reaction catalyzed by a diphosphomevalonate decarboxylase using diphosphomevalonate (A) or using a 3-hydroxyalk-4-enoate (B) as a substrate.
FIG. 2 shows the reaction catalyzed by a diphosphomevalonate decarboxylase using 3-hydroxypent-4-enoate as a substrate leading to the production of 1,3-butadiene.
FIG. 3 shows the reaction catalyzed by a diphosphomevalonate decarboxylase using 3-hydroxy-4-methylpent-4-enoate or 3-hydroxy-3 methylpent-4-enoate as a substrate leading to the production of isoprene.
FIG. 4 shows a scheme of the ADP quantification assay, monitoring NADH consumption by the decrease of absorbance at 340 nm.
FIG. 5 shows a mass spectrum of the enzymatic assay for 3-hydroxypent-4-enoate phosphorylation.
FIG. 6 shows a mass spectrum of the enzyme-free control assay for 3-hydroxypent-4-enoate phosphorylation.
FIG. 7 shows the plot of the velocity as a function of substrate concentration for the phosphotransferase reaction catalyzed by the mutant L200E of MDP decarboxylase from Th. acidophilum. Initial rates were computed from the kinetics over the 30 first minutes of the reaction.
FIG. 8 shows the production of 1,3-butadiene from 3-phosphonoxypent-4-enoate in the absence and presence of isoprene synthase from Pueraria montana var. lobata. 



Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation.
EXAMPLES
Example 1
Cloning, Expression and Purification of Enzymes
Cloning, Bacterial Cultures and Expression of Proteins.
The genes encoding studied enzymes were cloned in the pET 25b or pET 22b vectors (Novagen). A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. Competent E. coli BL21(DE3) cells (Novagen) were transformed with these vectors according to the heat shock procedure. The transformed cells were grown with shaking (160 rpm) on ZYM-5052 auto-induction medium (Studier F W, Prot. Exp. Pur. 41, (2005), 207-234) for 6 hours at 37° C. and protein expression was continued at 28° C. overnight (approximately 16 hours). The cells were collected by centrifugation at 4° C., 10,000 rpm for 20 min and the pellets were frozen at −80° C.
Protein Purification and Concentration.
The pellets from 200 ml of culture cells were thawed on ice and resuspended in 5 ml of Na2HPO4 pH 8 containing 300 mM NaCl, 5 mM MgCl2 and 1 mM DTT. Twenty microliters of lysonase (Novagen) were added. Cells were incubated 10 minutes at room temperature and then returned to ice for 20 minutes. Cell lysis was completed by sonication for 3×15 seconds. The bacterial extracts were then clarified by centrifugation at 4° C., 10,000 rpm for 20 min. The clarified bacterial lysates were loaded on PROTINO-1000 Ni-TED column (Macherey-Nagel) allowing adsorption of 6-His tagged proteins. Columns were washed and the enzymes of interest were eluted with 4 ml of 50 mM Na2HPO4 pH 8 containing 300 mM NaCl, 5 mM MgCl2, 1 mM DTT, 250 mM imidazole. Eluates were then concentrated and desalted on Amicon Ultra-4 10 kDa filter unit (Millipore) and resuspended in 0.25 ml 50 mM Tris-HCl pH 7.5 containing 0.5 mM DTT and 5 mM MgCl2. Protein concentrations were quantified by direct UV 280 nm measurement on the NanoDrop 1000 spectrophotometer (Thermo Scientific). The purity of proteins varied from 70% to 90%.
Example 2
Characterization of the 3-hydroxypent-4-enoate Phosphorylation Activity
The release of ADP that is associated with 1,3-butadiene production from 3-hydroxypent-4-enoate, was quantified using the pyruvate kinase/lactate dehydrogenase coupled assay (FIG. 4). The purified mevalonate diphosphate decarboxylases from Th. acidophilum, Th. volcanium and S. mitis were evaluated for their ability to phosphorylate 3-hydroxypent-4-enoate releasing ADP.
The studied enzymatic reaction was carried out under the following conditions at 37° C.:

    50 mM Tris-HCl pH 7.5
    10 mM MgCl2 
    100 mM KCl
    5 mM ATP
    0.4 mM NADH
    1 mM Phosphoenolpyruvate
    3 U/ml Lactate dehydrogenase
    1.5 U/ml Pyruvate kinase
    50 mM 3-Hydroxypent-4-enoate

The pH was adjusted to 7.5



Each assay was started by the addition of a particular enzyme (at a concentration from 0.05 to 1 mg/ml) and the disappearance of NADH was monitored by following the absorbance at 340 nm. Assays with mevalonate diphosphate (MDP) decarboxylases gave rise to a reproducible increase in ADP production in the presence of 3-hydroxypent-4-enoate. The enzymes from the Thermoplasma phylum displayed higher phosphotransferase activity than decarboxylase from S. mitis (Table 3).








TABLE 3





Mevalonate diphosphate decarboxylase
Activity, nmol/min/mg protein
















Th. acidophilum (mutant L200E)
138


Th. volcanium
114


S. mitis
0.52









Mass spectrometry was then applied to confirm the formation of 3-phosphonoxypent-4-enoate in the assay with mutant L200E of MDP decarboxylase from Th. acidophilum. 
Example 3
Mass Spectrometry Analysis of the Phosphorylation Reaction
The desired enzymatic reactions were carried out under the following conditions:

    50 mM Tris-HCl pH 7.5
    10 mM MgCl2 
    10mM KCl
    20 mM 3-hydroxypent-4-enoate
    20 mM ATP
    0.2 mg/ml purified MDP decarboxylase from Th. acidophilum (mutant L200E).


The control reactions without enzyme, without substrate and without ATP were run in parallel. The assays were incubated overnight without shaking at 37° C. Typically, an aliquot of 200 μl reaction was removed, centrifuged and the supernatant was transferred into a clean vial. The MS spectra were obtained on Esquire 3000 (Bruker) Ion Trap Mass Spectrometer with Electrospray Ionization Interface in negative ion mode. The presence of 3-phosphonoxypent-4-enoate was evaluated. MS analysis showed an [M-H]− ion at m/z 194.9 corresponding to 3-phosphonoxypent-4-enoate from the sample containing the enzyme but not from the negative controls (FIGS. 5, 6).
Example 4
Kinetic Parameters of Reaction of 3-hydroxypent-4-enoate Phosphorylation
Kinetic parameters were determined by varying substrate concentration between 0 and 30 mM under assay conditions, described in example 2.
FIG. 7 shows an example of a Michaelis-Menten plot corresponding to the data collected for the mutant L200E of MDP decarboxylase from Th. acidophilum. This enzyme was found to have a KM of 3.7 mM and a kcat of 0.09 sec−1.
Example 5
Butadiene production from 3-hydroxypent-4-enoate
The desired enzymatic reaction was carried out under the following conditions:

    50 mM Tris HCl pH 7.5
    10 mM MgCl2 
    20 mM KCl
    50 mM ATP
    200 mM 3-hydroxypent-4-enoate

The pH was adjusted to 7.5



Each assay was started by the addition of a particular enzyme to 0.5 ml of reaction mixture. The assays were then incubated with shaking at 37° C. in a 2 ml sealed vial (Interchim). Control reactions were run in parallel. After 48 hours of incubation the reaction mixtures were analyzed as follows. To 0.5 mL of each sample, 0.125 ml of heptane were added and the sample was incubated at 25° C. for 1 hour with shaking. The upper heptane phase was analyzed by gas chromatography (GC) on a Varian 430-GC chromatograph equipped with a FID detector. A 1 μL sample was separated on the GC using an Rt-Alumina BOND/Na2SO4 column (Restek) and nitrogen carrier gas. The oven cycle for each sample was 130° C. for 10 minutes, increasing temperature at 20° C./minute to a temperature of 200° C., and a hold at 200° C. for 10 minutes. The total run time was 23.5 minutes The enzymatic reaction product was identified by comparison with commercial 1,3-butadiene (Sigma). The results of butadiene production are presented in Table 4.








TABLE 4






1,3-Butadiene peak area,


Assay
arbitrary units
















Without substrate
0


Without enzyme
0.8


With 11 mg/ml of purified
1.0


S. mitis MDP decarboxylase


Combining assay with 1 mg/ml
1.6


Th. acidophilum enzyme (mutant L200E)


and 10 mg/ml S. mitis enzyme









The formation of 1,3-butadiene observed in the assay without enzyme is probably due to the spontaneous decomposition of 3-hydroxypent-4-enoate. The addition of MDP decarboxylase from S. mitis led to a 1.25-fold increase of butadiene production after 48 h of incubation. The highest production of isobutene was observed in the assay combining the MDP decarboxylase from Th. acidophilum and the MDP decarboxylase from S. mitis. This indicated that the two enzymes present in the assay were performing complementarily the two steps of reaction producing butadiene from 3-hydroxypent-4-enoate: transfer of the terminal phosphoryl group from ATP to the C3-oxygen of 3-hydroxypent-4-enoate followed by combined dephosphorylation-decarboxylation of the intermediate 3-phosphonoxypent-4-enoate.
Example 6
Butadiene Production from 3-phosphonoxypent-4-enoate
3-phosphonoxypent-4-enoate is synthesized by company specialized in custom synthesis, Syntheval (France).
The studied enzymatic reactions are carried out under the following conditions at 37° C.:

    50 mM Tris-HCl pH 7.5
    10 mM MgCl2 
    0-20 mM KCl
    5 mM ATP
    0-250 mM 3-phosphonoxypent-4-enoate

The pH is adjusted to 7.5



The reaction is initiated by the addition of a particular enzyme to 0.5 ml of reaction mixture. The free-enzyme control reactions are carried out in parallel. The assays are incubated with shaking at 37° C. in a 2 ml sealed vial (Interchim). The production of butadiene is measured by analyzing aliquots sampled over a 72 hour incubation period. Volatile compounds in the headspace of reaction mixture are collected and directly injected into a Varian 430-GC chromatograph equipped with a flame ionization detector and an Rt-Alumina BOND/Na2SO4 column (Restek). Additionally, 1,3-butadiene production is monitored by analysis of reaction mixture using gas chromatography as described in example 5. Commercial 1,3-butadiene is used as reference.
Example 7
Characterization of the 3-hydroxy-3-methylpent-4-enoate Activity
The release of ADP associated with isoprene production from 3-hydroxy-3-methylpent-4-enoate is quantified using the pyruvate kinase/lactate dehydrogenase coupled assay (FIG. 4). The purified mevalonate diphosphate decarboxylases are evaluated for their ability to phosphorylate this substrate releasing ADP.
The studied enzymatic reactions are carried out under the following conditions at 37° C.:

    50 mM Tris-HCl pH 7.5
    10 mM MgCl2 
    100 mM KCl
    5 mM ATP
    0.4 mM NADH
    1 mM Phosphoenolpyruvate
    3 U/ml Lactate dehydrogenase
    1.5 U/ml Pyruvate kinase
    50 mM 3-Hydroxy-3-methylpent-4-enoate


Each assay is started by the addition of a particular enzyme (at a concentration from 0.05 to 1 mg/ml) and the disappearance of NADH is monitored by following the absorbance at 340 nm.
Example 8
Characterization of the 3-hydroxy-4-methylpent-4-enoate Phosphorylation Activity
The release of ADP associated with isoprene production from 3-hydroxy-4-methylpent-4-enoate is quantified using the pyruvate kinase/lactate dehydrogenase coupled assay (FIG. 4). The purified mevalonate diphosphate decarboxylases are evaluated for their ability to phosphorylate this substrate releasing ADP.
The studied enzymatic reactions are carried out under the following conditions at 37° C.:

    50 mM Tris-HCl pH 7,5
    10 mM MgCl2 
    100 mM KCl
    5 mM ATP
    0.4 mM NADH
    1 mM Phosphoenolpyruvate
    3 U/ml Lactate dehydrogenase
    1.5 U/ml Pyruvate kinase
    50 mM 3-Hydroxy-4-methylpent-4-enoate


Each assay is started by the addition of a particular enzyme (at a concentration from 0.05 to 1 mg/ml) and the disappearance of NADH is monitored by following the absorbance at 340 nm.
Example 9
Isoprene Production from 3-hydroxy-3-methylpent-4-enoate
The studied enzymatic reactions are carried out under the following conditions at 37° C.:

    50 mM Tris-HCl pH 7.5
    10 mM MgCl2 
    20 mM KCl
    5 mM ATP
    0-200 mM 3-hydroxy-3-methylpent-4-enoate

The pH is adjusted to 7.5



The reaction is initiated by the addition of one or two particular enzyme(s) to 0.5 ml of reaction mixture. The enzyme-free control reactions are carried out in parallel. The assays are incubated with shaking at 37° C. in a 2 ml sealed vial (Interchim). The gas present in the headspace is collected and analyzed by gas chromatography coupled with a flame ionization detector. The enzymatic reaction product is identified by comparison with commercial isoprene (Sigma).
Example 10
Isoprene Production from 3-hydroxy-4-methylpent-4-enoate
The studied enzymatic reactions are carried out under the following conditions at 37° C.:

    50 mM Tris-HCl pH 7.5
    10 mM MgCl2 
    20 mM KCl
    5 mM ATP
    0-200 mM 3-hydroxy-4-methylpent-4-enoate

The pH is adjusted to 7.5



The reaction is initiated by the addition of one or two particular enzymes to 0.5 ml of reaction mixture. The enzyme-free control reactions are carried out in parallel. The assays are incubated with shaking at 37° C. in a 2 ml sealed vial (Interchim). The gas present in the headspace is collected and analyzed by gas chromatography coupled with a flame ionization detector. The enzymatic reaction product is identified by comparison with commercial isoprene (Sigma).
Example 11
1,3-butadiene Production from 3-phosphonoxypent-4-enoate by Using a Terpene Synthase
The sequence of the isoprene synthase inferred from the genome from Pueraria montana var. lobata (Uniprot Q6EJ97) was generated by oligonucleotide concatenation to fit the codon usage of E. coli. The amino acid sequence of the enzyme is shown in SEQ ID NO: 30. A stretch of 6 histidine codons was inserted after the methionine initiation codon to provide an affinity tag for purification. The gene thus synthesized was cloned in a pET 25b(+) expression vector (the vector was constructed by GeneArt AG). The corresponding enzyme was expressed in E. coli and purified as described in Example 1.
The reactions were performed in sealed vials. The total volume was 0.5 ml. Final concentrations were 5 mg/ml enzyme, 50 mM 3-phosphonoxypent-4-enoate, 4 mM DTT, 50 mM MgCl2, 50 mM KCl, 50 mM Tris-HCl buffer pH 7.5. The incubation was carried out at 37° C. for 24 h. The control reactions without enzyme or without substrate were performed in parallel under the same conditions.
One ml of the gaseous phase of the reaction was collected and analyzed by Gas-Chromatography with Flame Ionization Detector (GC-FID) ((Brucker GC 450) using a RTX-alumina column (Varian), with an isocratic elution at 130° C. and nitrogen as carrier gas at flow rate of 1.5 ml/min. The retention of commercial 1,3-butadiene (Sigma) in these conditions was 7.4 min.
Results: No formation of 1,3-butadiene was observed without substrate. The GC analysis of reactions without enzyme and with non-relevant enzyme showed only traces of butadiene resulted from the thermal decomposition of the 3-phosphonoxypent-4-enoate. The catalytic tests showed a significant increase of butadiene production in the presence of purified isoprene synthase from Pueraria montana var. lobata. The ratio of butadiene produced after 24 hours incubation in the presence of isoprene synthase versus butadiene produced in the absence of enzyme is about 5 fold judging from butadiene peak areas (FIG. 8). These results clearly indicate that a terpene synthase such as isoprene synthase catalyzes the conversion of a 3-phosphonoxyalk-4-enoate to a 1,3-diene, in particular 3-phosphonoxypent-4-enoate to 1,3-butadiene.

## Claims

1. A method for the production of a 1,3-diene compound that comprises converting a 3-hydroxyalk-4-enoate with a diphosphomevalonate decarboxylase (EC 4.1.1.33) into a 1,3-diene compound, wherein the 3-hydroxyalk-4-enoate has the general formula of Cn+1H2nO3 with 3<n<7 and comprises a 3-hydroxypent-4-enoate as a common motif and optionally a methyl substitution on carbon 3 and carbon 4.

2. The method of claim 1 wherein the 3-hydroxyalk-4-enoate is 3-hydroxypent-4-enoate and the produced 1,3-diene compound is 1,3-butadiene.

3. The method of claim 1 wherein the 3-hydroxyalk-4-enoate is 3-hydroxy-4-methylpent-4-enoate or 3-hydroxy-3-methylpent-4-enoate and the produced 1,3-diene compound is isoprene.

4. The method of claim 1 wherein the diphosphomevalonate decarboxylase comprises the amino acid sequence of SEQ ID NOs: 1 to 19 or 22 to 29.

5. The method of claim 4 wherein the diphosphomevalonate decarboxylase comprises the amino acid sequence of SEQ ID NO: 6, 16, 17, 18 or 19.

6. The method of claim 1, wherein
(i) a first diphosphomevalonate decarboxylase converts the 3-hydroxyalk-4-enoate into the corresponding 3-phosphonoxyalk-4-enoate; and
(ii) a second diphosphomevalonate decarboxylase being different from the first diphosphomevalonate decarboxylase which converts said 3-phosphonoxyalk-4-enoate into said 1,3-diene compound by a decarboxylation reaction.

7. The method of claim 6 wherein the first diphosphomevalonate decarboxylase is a protein comprising the amino acid sequence as shown in SEQ ID NO: 18.

8. The method of claim 6 wherein the second diphosphomevalonate decarboxylase is a protein comprising the amino acid sequence as shown in SEQ ID NO: 24.

9. A method for the production of a 1,3-diene compound comprising:
(i) converting a 3-hydroxyalk-4-enoate into a 3-phosphonoxyalk-4-enoate by a disphosphomevalonate decarboxylase (EC 4.1.1.33); and
(ii) converting 3-phosphonoxyalk-4-enoate into a 1,3-diene compound by a terpene synthase; wherein 3-hydroxyalk-4-enoate has the general formula of Cn+1 H2nO3 with 3<n<7 and comprises a 3-hydroxypent-4-enoate as common motif and optionally a methyl substitution on carbon 3 and carbon 4.

10. The method of claim 9, wherein said terpene synthase is an isoprene synthase (EC 4.2.3.27).

11. A method for producing a 1,3-diene compound comprising enzymatically converting a 3-phosphonoxyalk-4-enoate into the corresponding 1,3-diene compound by a terpene synthase; wherein 3-hydroxyalk-4-enoate has the general formula of Cn+1 H2nO3 with 3<n<7 and comprises a 3-hydroxypent-4-enoate as common motif and optionally a methyl substitution on carbon 3 and carbon 4.

12. The method of claim 11, wherein said terpene synthase is an isoprene synthase (EC 4.2.3.27).

13. The method of claim 11 wherein the 3-phosphonoxyalk-4-enoate is 3-phosphonoxypent-4-enoate and the produced 1,3-diene is 1,3-butadiene.

14. The method of claim 11 wherein the 3-phosphonoxyalk-4-enoate is 3-phosphonoxy-4-methylpent-4-enoate or 3-phosphonoxy-3-methylpent-4-enoate and the produced 1,3-diene is isoprene.

15. The method of claim 1 which is carried out in vitro.

16. The method of claim 1 wherein a co-substrate is added.

17. The method of claim 16 wherein the co-substrate is ATP, a rNTP, a dNTP, a polyphosphate or pyrophosphate, or a mixture of any of these compounds.

18. The method of claim 1 wherein the method is carried out by making use of a microorganism producing said enzyme or said enzymes.

19. The method of claim 18, wherein the microorganism is capable of producing a 3-hydroxyalk-4-enoate and/or a 3-phosphonoxyalk-4-enoate.

20. The method of claim 6 wherein the first diphosphomevalonate decarboxylase is:
(A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 16;
(B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 17; or
(C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 19.

21. The method of claim 6 wherein the second diphosphomevalonate decarboxylase is:
(A) a protein comprising the amino acid sequence as shown in SEQ ID NO: 12;
(B) a protein comprising the amino acid sequence as shown in SEQ ID NO: 22;
(C) a protein comprising the amino acid sequence as shown in SEQ ID NO: 23;
(D) a protein comprising the amino acid sequence as shown in SEQ ID NO: 1;
(E) a protein comprising the amino acid sequence as shown in SEQ ID NO: 7;
(F) a protein comprising the amino acid sequence as shown in SEQ ID NO: 25;
(G) a protein comprising the amino acid sequence as shown in SEQ ID NO: 26;
(H) a protein comprising the amino acid sequence as shown in SEQ ID NO: 27;
(I) a protein comprising the amino acid sequence as shown in SEQ ID NO: 28; or
(J) a protein comprising the amino acid sequence as shown in SEQ ID NO: 29.

22. The method of claim 9, wherein said terpene synthase is a monoterpene synthase, an alpha-farnesene synthases (EC 4.2.3.46), a beta-farnesene synthase (EC 4.2.3.47), a myrcene/(E)-beta-ocimene synthase (EC 4.2.3.15) or a pinene synthase (EC 4.2.3.14).

23. The method of claim 11, wherein said terpene synthase is a monoterpene synthase, an alpha-farnesene synthases (EC 4.2.3.46), a beta-farnesene synthase (EC 4.2.3.47), a myrcene/(E)-beta-ocimene synthase (EC 4.2.3.15) or a pinene synthase (EC 4.2.3.14).

24. The method of claim 9 wherein the diphosphomevalonate decarboxylase comprises the amino acid sequence of SEQ ID NOs: 1 to 19 or 22 to 29.